1
INTRODUCTION
1. The urgency of the study
Gout is a common disease, due to the deposition of monosodium
urate (MSU) crystals in synovial fluid or tissues. Between 1 - 4% of
the adult population worldwide has gout. In developed countries, the
incidence of gout in men is between 3 - 6% of the population, and
between 1 - 2% of the population. In some developing countries, the
incidence of gout may increase to 10%. Gout usually develops in
stages. About 25% of gout patients have joint damage, irreversible
joint deformity. The quality of mental and physical life of gout
patients decreased, due to the frequent affects of acute gout attacks,
the number of swollen, painful joints increased over time, the
progression and spread of tophus, using colchicin, corticosteroids...
To research into new herbal medicine for gout treatment that can
limit unwanted effects is meaningful and necessary. Therefore, over
the past decade, scientists have studied a variety of herbal and
traditional medicines for the gout treatment. Vietnam traditional
medicine has many effective formulas in treating gout, Si Miao Yan
formula is one of them. The composition of Si Miao Yan formula
consists of Semen Coicis, Phellodendron sp Rutaceae, Achyranthes
bidenta, Rhizoma Atractylodis. Some experimental studies have
shown that polysaccharides in Rhizoma Atractylodis and baicalein,
oroxylin A in Cortex Oroxyli (replaced Phellodendron sp Rutaceae)
have anti-inflammatory effect. Stigmasterol and p-coumaric acid in
Semen Coicis are involved in the elimination of uric acid. We
adjusted the dosage, the drug mix, the drug form then evaluated the
effects of Tu dieu tan granule on two objectives:
2. Study objectives
1.
Evaluating the effectiveness of Tu dieu tan granule on acute
and sub-chronic toxicity, anti-inflammatory, analgesic and
lowering serum urate level in experimental.
2.
Evaluating anti-inflammatory, analgesic and lowering serum
urate level and unexpected effects of Tu dieu tan granule in
patient with chronic gout.
3.
Practical implications and new contributions of the thesis
This scientific study based on the effect evaluation of a new
herbal product called Tu dieu tan granule which was used in chronic
2
gout treatment. According to the clinical experience, we reduce the
traditional formula Si Miao to suit Vietnamese climate, physiological
characteristics of the people and the purpose of treatment: replacing
Phellodendron sp Rutaceae and Achyranthes bidenta with Cortex
Oroxyli and Radix Achyranthes asperae, changing the dose of Si
Miao formula, changing to granule form. We had given the scientific
evidence to explain the anti-inflammatory, analgesic and hypouricemic
effects of Tu dieu tan granule in experimental and clinical.
The experimental results showed that Tu dieu tan granule has not
shown the acute and subchronic toxicity. Tu dieu tan granule has
anti-inflammatory, analgesic and hypouricemic effects by excreting
uric acid through urinary system in the experimental model. After 30
days of treatment, the clinical results showed that the combined of Tu
dieu tan granule with allopurinol reduced serum uric acid (SUA)
levels were 200,42 ± 100,14μmol/l; the effectiveness of lowering
SUA decreased by 98,33%; decreased the number of swollen, painful
joints, improved mean pain intensity on VAS 1 scale and physical
activities function according to VAS2, VAS3. Improving median
function assessed by HAQ was 0,96 ± 0,32 point; Its efficacy of
improving traditional symptoms was 98,33%. The improvement of
these indicators in the study group were statistically significantly
higher compared to the control group (p < 0,05). Tu dieu tan granule
had effect in treating both the wind damp heat impediment pattern
and the phlegm blood stagnation pattern with SUA levels decreased
222,26 ± 114,84μmol/l; 194,97 ± 96,69μmol/l and Nimodiping score
improvement was 10,92 ± 2,31 points; 10,62 ± 2,83 points; the
difference between two patterns was not statistically significantly. No
adverse effects of Tu dieu tan granule in clinical or subclinical were
observed. These findings are scientific indications of the safety, antiinflammatory and analgesic, hypouricemic effects of Tu dieu tan
granule in treating chronic gout.
4.Thesis structure. Apart from the Introduction and Conclusions
chapters, the thesis consists of four chapters.
Chapter 1: Literature review
35 pages
Chapter 2: Study subjects and methods
25 pages
Chapter 3: Results
37 pages
Chapter 4: Discussion
34 pages
And 38 tables, 6 graphs, 5 images, 13 appendixes, and 151
references (34 Vietnamese references, 93 English references, 24
Chinese references).
3
Chapter 1
LITERATURE REVIEW
1.1. The concept of gout according to modern medicine
1.1.1. Cause and pathogenesis
The pathogenesis of gout is due to hyperuricemia. When SUA
level rises over saturation, uric acid deposits in the form of MSU in
joint or tissues, triggering inflammatory reactions that relating acute
gout attacks. Between gout flares, MSU are deposited and formed
tophus. The more uric acid is deposited the more inflammatory reaction
becomes a continuous inflammation circle.
1.1.2. Diagnosis of chronic gout. According to Bennett and Wood
criteria (1968).
1.1.3. Treatment of chronic gout
Besides reducing the symptoms of pain and inflammation, the most
important target is reducing SUA, which allows patients to prevent
complications of chronic renal failure, organ damage. Treatment Principle:
Patients maintain a diet of chronic gout, combined with hypouricemic
drug. Start at low doses, gradually increasing to therapeutic doses and
maintain continuously, uninterrupted, which can be combined with
NSAIDs or colchicin to prevent acute gout attacks. Target is lowering
SUA levels less than 6mg/dl (360μmol/l) in chronic gout or 5mg/dl
(300μmol/l) in chronic gout with tophus. Common drug are xanthin
oxidase inhibitors (allopurinol, febuxostat ... ) which inhibit xanthin
oxidase. According to American College of Rheumatology, starting
dose of allopurinol should be no greater than 100mg/day and start at
50mg/day in stage 4 or severe CKD. Dosage can be raised above
300mg daily, even with renal impairment. Febuxostat is indicated for
patients with allopurinol-resistant or allergic allopurinol. Febuxostat
inhibits vasoactive agents and prevents inflammation. In addition,
other urate lowering therapy such as uricosuric therapy (probenecid,
benzbromarone), uricolytic drugs (Uricozym, Pegloticase), urine
alkalinization (sodium bicarbonate solution) may be considered.
1.2. The concept of gout according to traditional medicine
1.2.1. Cause and pathogenesis
According to traditional medicine, gout has the name is “Tong Feng”.
Tong Feng is used to refer the pain causes by wind. Gout is cateogrised as
“Tong bi” according to traditional medicine (Bi means standstill, not
cleared). The causes of “Tong bi” include six external climatic factors,
seven emotional factors, and other pathogenic factors.
4
1.2.2. Treatment of gout
Depending on the clinical types, there are appropriate treatments
principles and specific formulas. According to Hao Xian Gou and Jie
Tian Ping – China (2008), Tong Feng is divided into five patterns:
Wind cold damp impediment pattern, wind damp heat impediment
pattern, phlegm blood stagnation pattern, spleen and kidney yang
deficiency pattern, liver and kidney yin deficiency pattern. For the
wind cold damp impediment pattern, the principles include of
dispelling wind, scattering cold, eliminating dampness, circulating
meridians; the appropriate herbal formula are Tigao Tai Tang or
Fangjian Fengge Tang. For the wind damp heat impediment pattern,
the principles include of dispelling wind, clearing heat, eliminating
dampness; the appropriate herbal formula are Er Miao San combined
with Bai Hu Gui Zhi Tang; for the damp heat pattern include
numbness, the principles include of clearing heat, eliminating
dampness, circulating meridians, analgesic, the appropriate herbal
formula are Si Miao San combined with Long Dan Da Gan Tang. For
the phlegm blood stagnation pattern, the principles include of
circulating blood, dispelling stasis, transforming phlegm; the
appropriate herbal formula are Fu Yuan Huo Xue Tang combined
with Er Chen Tang. For the spleen and kidney yang deficiency
pattern, the principles include of tonifying spleen qi, supplementing
kidney yang; the appropriate herbal formula is Wu Tou Gui Zhi
Tang. For the liver and kidney yin deficiency pattern, the principles
include of nourishing yin, supplementing kidney and liver yin; the
appropriate herbal formula is Du Huo Ji Sheng Tang.
1.3. Review on experimental models evaluating toxicity and the
effectives of herbal medicine in treating gout.
Tu dieu tan granule is derived from the traditional formula Si
Miao. Although the formula composition does not change, but due to
the reduction of each dose in the formula compared to the old and
converted formulation, Tu dieu tan granule should be determined
toxicity to ensure safety for testing the next phase. Tu dieu tan granule
was evaluated acute and sub-chronic toxicity. It was evaluated antiinflammatory, analgesic and hypouricemic effects in experimental
models such as the carrageenin-induced rat paw oedema, rat peritonitis
inflammatory, rat arthritis after intrasynovial injection of sodium urate;
hot plate, hyperalgesia to thermal stimulation in rat by the Thermal
Plantar Test Instrument; potassium oxonat induced hyperuricemia in
5
rat, diuretic and uricosuric activity in mice, xanthin oxidase inhibatory
activity in vitro and in vivo models.
1.2. Review on gout treatment studies
1.2.1. Review on gout treatment studies according
to Western medicine
Currently, almost studies have focused on reducing SUA levels and
maintaining SUA levels below 6mg/dL (<360μmol/L). Nextgeneration drugs were being clinical used: febuxostat; 3,4-dihydroxy5-nitrobenzaldehyde (DHNB)… In addition, based on the gout flare
mechanism, researchers was still testing IL-1β inhibitors (canakinumab,
anakinra) on anti-inflammatory and analgesic effects in gout flares. In
Vietnam, there has been no research on new medicine or gout treatment
medication in the last decade. Most studies of Western medicine focused
on the medicine abuse (corticosteroid). Some studies assessed the
knowledge of patients in preventing and using gout medicines.
1.2.2. Review on gout treatment studies according
to traditional medicine
In experimental, many models were built up to evaluate the
effectiveness of gout medication on anti-inflammatory, analgesic, and
lowering SUA level such as: Xanthin oxidase inhibatory in vitro and in
vivo models, rat arthritis after intrasynovial injection of sodium urate,
evaluating hypouricemic effects in potassium oxonat induced
hyperuricemia in rat... Shen Wei Zeng, Pang Xue Feng used rat arthritis
after intrasynovial injection of sodium urate in evaluating antiinflammatory and analgesic effects of Dang Gui Nian Tong Tang, Zhi
Tong Qu Feng Tang. In clinical, many scientists have built up and
studied the effectiveness of new herbal formulas such as: HA1 herbal
formula, GLP hypouricaemia... Other authors have investigated the
association between Western medicine and traditional medicine in order
to have higher therapeutic efficacy than either Western medicine or
traditional medicine. All researchs have the same procedure is controlled
trial, comparison study (Studying on the effectiveness of Tong Feng
Wan, or the combined of Narcaricin and Si Wu Tang).
1.3. Review of Tu dieu tan granule
1.3.1. Origin and traditional efficacy
Tu dieu tan granule is derived from the traditional medicine Si Miao
in Fang Ji Xue book, with changing doses and replacing Phellodendron
sp Rutaceae and Achyranthes bidenta with Cortex Oroxyli and Radix
Achyranthes asperae. The increased dosage of Tu dieu tan granule was
6
intended to enhance the effectiveness on excreting phlegm, eliminating
dampness, clearing heat, circulating blood.
1.3.2. Components and efficacy of Tu dieu tan granule
- Tu dieu tan granule 7,5g with the composition of 1,13g dried extract
Radix Achyranthes asperae; 0,56g dried extract Cortex Oroxyli; 1,5g
dried extract Semen Coicis; 0,56g dried extract Rhizoma Atractylodis;
excipients enough for 7,5g.
- Semen Coicis has effective on tonifying spleen qi, diuretic.
Chemical composition: Semen Coicis contains starch, protein, amino
acid. Substances isolated from the semen include coixenolid,
policosanol, phytosterols, lactams, phenol compounds. P-coumaric
acid in Semen Coicis granule inhibits the nucleation, growth and
aggregation of stone crystal, inhibits the binding of crystals on the
renal tubular epithelium, inhibitits the formation of calcium oxalate
crystals which depends on acid p-coumaric concentration.
- Radix Achyranthes asperae has effective on circulating blood,
stimulating meridians, diuretic, nourishing liver and kidney,
dispelling wind dampness. Chemical composition: Betaine,
achyrathine, β-ecdysone, stigmasterol. Radix Achyranthes asperae
contains saponin, polysaccharide, ketosteroid and β-sitosterol.
Saponin in Radix Achyranthes asperae has an inhibitory effect on
cytokines, which reduces swelling and arthritis. Achyranthine has
diuretic effect in white mice and does not change the pH in urine.
- Cortex Oroxyli has effective on clearing heat, detoxifying,
eliminating dampness in lower burner. Chemical composition:
Baicalein, oroxylin A, chrysin, tetuin, β-sitosterol, tannic acid.
Cortex Oroxyli contains p-coumaric acid. Baicalein, oroxylin A in
Cortex Oroxyli has acute anti-inflammatory effects by inhibiting NFκB, thereby inhibiting the release of inflammatory response factors
PGE2, IL6, IL-1β.
- Rhizoma Atractylodis has effective on scattering cold and
circulating cuticle, tonifying spleen qi, detoxifying, eliminating
phlegm. Chemical composition: Mostly are hinesol or β-eudesmol,
atractylon. Polysaccharide has effective on regulating the intestine
immune system against candida albicans. β-eudesmol and atractylon
protect the liver cells.
7
Chapter 2
STUDY MATERIALS, SUBJECTS AND METHODS
2.1. Study materials
2.1.1. Baseline medicines
- Hypouricemic medicine: Allopurinol oral tablet contains 300mg
allopurinol, excipients just enough. This product is distributed by
Domesco Medical Import Export Joint Stock Corporation.
- NSAIDs: Mofen oral tablet contains 400mg ibuprofen, excipients
just enough. This product is distributed by Medopharm – India.
2.1.2. Studied medicines
- Tu dieu tan granule 7,5g with the composition of 1,13g dried extract
Radix Achyranthes asperae; 0,56g dried extract Cortex Oroxyli; 1,5g
dried extract Semen Coicis; 0,56g dried extract Rhizoma Atractylodis;
excipients enough for 7,5g. Tu dieu tan granule is produced by
Department of Pharmacy/Military Institute of Traditional Medicine
(MITM), has been granted certification basic-level standard.
- Placebo: Presenting format like Tu dieu tan granule. One package
with the composition of 7,5g lactose, excipients enough. Placebo is
produced by Department of Pharmacy/Military Institute of Traditional
Medicine.
2.2. Study subjects
The studies were conducted from March 2015 to May 2017.
2.2.1. Subjects in experimental studies: Swiss mice, Wistar rats, both
species are healthy, qualify for participation in experimental models.
Location of the experimental studies: Department of Pharmacology/Hanoi
Medical University, Experimental Laboratory/MITM, Department of
Pharmacology/Hanoi University of Pharmacy.
2.2.2. Subjects in clinical studies: The sample size was calculated
according to formulas for clinical studies established by World
Health Organisation. The study was performed on 120 inpatients at
MITM, qualify the clinical inclusion and exclusion criterias:
* Inclusion criteria: Selecting patients regardless of age, gender,
occupation, residence, consent to participate in the study, based on the
criteria of Western medicine and traditional medicine. According to
Western medicine: Patient with chronic gout (include gout flare) was
diagnosed according to Bennett and Wood criteria (1968), with SUA
levels > 420μmol/l for male and > 360μmol/l for female. According to
traditional medicine: Patient was diagnosed wind damp heat
impediment pattern or phlegm blood stagnation pattern.
8
* Exclusion criteria: Subjects with hepatic failure, renal failure,
myocardial infarction, cerebral hemorrhage; Long-term gastrointestinal
disturbances affect the process of drug absorption and metabolism; mental
illness, malnutrition, infectious diseases; contraindications for allopurinol;
Tong Feng symptoms do not belong to the wind damp heat impediment
pattern or phlegm blood stagnation pattern; uncooperative in the studies;
pregnant or lactating women.
2.3. Study methods
2.3.1. Experimental studies: We conducted an experimental
controlled study with the following stages.
2.3.1.1. Investigating the acute and sub-chronic toxicity effects
* Acute toxicity: was assessed in white mice using Litchfield Wilcoxon method based on OECD and WHO guidelines. The mice
received the medication under investigation with increasing doses
through oral use. The aim was to identify the non-lethal highest dose
(0%), the lethal lowest dose (100%) and the intermediate doses. The
mice were observed for 72 hours to monitor their mortality rate and
overall health condition; and were observed for 7 days since
medication administration to monitor mortality rate.
* Sub-chronic toxicity: According to WHO guideline, Wistar rat
were devided into 3 groups: Controll group (distilled water);
Treatment group 1 (Tu dieu tan granule 1,8g/kg/day - corresponding
to the clinical dose); Treatment group 2 (Tu dieu tan granule
5,4g/kg/day - three times higher than the clinical dose). Rats were
drunk water and Tu dieu tan granule for 8 weeks continuously. The
rats were observed to monitor their weight, eating, sleeping, activity,
digestion, hematology, biochemistry, liver and kidney function, liver
and kidney histopathology. Comparisons were made between preand post-treatment, and between the treatment and control groups.
2.3.1.2. Acute anti-inflammatory effect
Evaluating anti-inflamatory effect in experimental models such as the
carrageenin-induced rat paw oedema, rat peritonitis inflammatory, rat
arthritis after intrasynovial injection of sodium urate. The carrageenininduced rat paw oedema model was conducted according to Winter
method. The rat peritonitis inflammatory model was conducted according
to Patel method. The rat arthritis after intrasynovial injection of sodium
urate model was conducted according to Faires and McCarty method.
2.3.1.3. Analgesic effect
9
Evaluating analgesic effect in experimental models such as the
hot plate model (evaluation the central analgesic effect), the
hyperalgesia to thermal stimulation in rat by the Thermal Plantar Test
Instrument (evaluation the peripheral analgesic effect). The hot plate
model was conducted according to Vogel method. The hyperalgesia
to thermal stimulation in rat by the Thermal Plantar Test Instrument
was conducted according to Randall-Selitto method.
2.3.1.4. Hypouricemic effect
Evaluating hypouricemic effect in experimental models such as the
potassium oxonat induced hyperuricemia in rat (evaluating hypouricemic
effect), diuretic and uricosuric activity in mice (evaluating the urinary uric
acid excretion), the xanthin oxidase inhibatory activity in vitro and in vivo
models (evaluating the synthesis inhibition effect of uric acid). The
potassium oxonat induced hyperuricemia in rat was conducted according
to Starvic method. The xanthin oxidase inhibatory activity in vitro and in
vivo model was conducted according to Nguyen Thi Thanh Mai method.
The diuretic and uricosuric activity in mice was conducted according to
Starvic method.
2.3.2. Clinical studies
Study design: Clinical intervention, prospective placebo-controlled
double-blind study.
2.3.2.1. Study procedure
After clinical examination, the patients who met the inclusion
criteria were enrolled into the study. An independent study staff
divided the study subjects into 2 groups, each group has 60 patients.
Patients were comparable regarding age, sex, disease level (the
number of painful and swollen joints, SUA level), traditional pattern.
Each patient’s identity was encoded using a code attached to his/her
medical record. The information whether the patients were in the
study or control group was entirely unknown to all the patients and
healthcare staff who were directly involved in the treatment of the
patients. The medication under investigation and the placebo were
presented in the same format.
During 30 treatment days, patients in the study group used
allopurinol and Tu dieu tan granule, patients in the control group
used allopurinol and placebo. Patient was treated on the first day of
the study (D0): Allopurinol (01 tablet taken once per day, after meal);
Tu dieu tan granule (1 package per time taken twice per day, 1 hour
before meal); placebo (1 package per time taken twice per day, 1
hour before meal). During the study, patients did not use other
medicines. In case of patient with gout flare was painful and ask for
10
analgesic medicine, he/she would be allowed to use Mofen 400mg in
5 days maximum (01 tablet per time, taken twice or three times per
day). The patients’ identities were only revealed after the course of
treatment was completed, then data collection, analysis and report
were carried out.
2.3.2.2. Study indicators and evaluation methods
- Describe characteristics of the study subjects: Age, gender,
historical symptoms (disease level, risk factors, traditional patterns).
- Clinical indicators: Study indicators was used to evaluate the
effectiveness of Tu dieu tan granule in clinical and subclinical and
compare the effectiveness between the control and study group, preand post-treatment. These indicators were assessed in the first day
(D0) and the 30th day (D30) of the treatment course: Anti-inflammatoy
effect (reduced the number of swollen joints); analgesic effect
(reduced the number of painful joints, improved VAS 1 scores);
improved active function (improved VAS2, VAS3, HAQ scores);
improved traditional symptoms according to Nimodiping score.
Evaluating the treatment effective in sub-clinical (D0, D30); evaluating
SUA levels reduction in the study and control groups, in two
traditional patterns.
- Unexpected effects were monitored during the whole course of
treatment: The unexpected symptoms in clinical; hematological
indicators in subclinical.
2.3.2.3. Statistical analyses: Data analyses were performed using SPSS
22.0. A p-value of ≤ 0.05 was considered statistically significant.
2.3.2.4. Research ethics: The study was approved by the Medical
Ethics Committee of Hanoi Medical University. Permission for the
study to be conducted at MITM was retrieved from the hospital’s
General Sciences Council.
Chapter 3
STUDY RESULTS
3.1. Results of experimental studies
3.1.1. Investigating the acute and sub-chronic toxicity effects
* Acute toxicity: After oral administration of Tu dieu tan granule
from 4 to 6 hours, all rats showed normal function, agility, smooth
fur, normal eating activities and dry stool. Rats drank Tu dieu tan
granule at the dose 62,5g and 78,1g were diarrhea on the last 2 days.
There were no adverse symptoms, no rats died within 72 hours of
administration and for 7 days. Acute toxicity and LD 50 of Tu dieu tan
granule have not been determined.
11
* Subchronic toxicity: During the experiments, the rats in all 3
groups showed normal function. There were no difference in overall
conditions and weight. Tu dieu tan granule at the dose of 1,8g/kg
after 8 weeks of continuous administration and 5,4g/kg after 4 weeks
of continuous administration showed no haematopoietic toxicity and
the treatment did not change the rats’ liver and kidney function
indicated by biochemiscal as well as histopathological tests. Tu dieu
tan granule at the dose of 5,4g/kg after 8 weeks of continuous
administration showed the changes in hematological indicators but
still in normal standards.
3.1.2. Acute anti-inflammatory effects
* The carrageenin-induced rat paw oedema model: Tu dieu tan
granule at the dose of 1,8g/kg and 5,4g/kg had no anti-inflammatory
effect at all times after causing inflammation.
* The rat peritonitis inflammatory model
Table 3.1. Acute anti-inflammatory effects of Tu dieu tan granule on
the volume and components of rat peritonitis inflammatory
Tu dieu tan Tu dieu tan
Biological
Aspirin
control
granule
granule
Indicators
200mg/kg
group
(n = 10)
(n = 10)
1,8g/kg
(n = 10)
5,4g/kg
(n = 10)
Volume of
peritonitis
1,33 ±
1,53 ±
inflammatory 2,81 ± 0,79
1,32± 0,79***
0,76***
0,38***
fluid
(ml/100g)
Number of
66,57±45,6 16,43±12,38* 21,96±13,47* 21,55±10,60*
white blood
0
*
*
*
cells (G/l)
Protein
content of
37,63±16,4 15,12±9,12**
16,19 ±
20,20 ±
peritonitis
8
*
8,57**
11,10*
fluid (mg/dl)
** p ≤ 0,01 and *** p ≤ 0,001 compared to control group
The results showed that Tu dieu tan granule at the two doses
reduced the volume and protein content of rat peritonitis
inflammatory statistically significantly compared to the biological
12
control group (p < 0,001), reduced the number of white blood cells (p
< 0,01); the effect was equivalent to aspirin 200mg/kg.
* The rat arthritis after intrasynovial injection of sodium urate
- The effects of Tu dieu tan granule on the inflammatory levels, size
and temperature of the knee joint: Tu dieu tan granule at the dose of
2,1g/kg had effect on reducing knee oedema, synovial fluid, lowering
the temperature of the inflammatory site, reducing the infiltration of
inflammatory cells after 6 hrs and 24 hours. The difference was
statistically significantly compared to the biological group (p < 0,05).
- Knee pathology in rats treated Tu dieu tan granule: The synovial
fluid has a sloughed area which is easy to observe. The synovial fluid
had less oedema fluid, urate crystals and infiltration of inflammed
cells than the biological control group.
1
2
1
2
(HE x 40)
(HE x 400)
Picture 3.1. Knee pathology in rats treated Tu dieu tan granule
(Rat number 25, treated Tu dieu tan granule)
1. The synovial fluid had less edema fluid, urate crystals
2. The infiltration of inflammed cells
3.1.3. Analgesic effects
* The hot plate model: Tu dieu tan granule at the dose of 3,6g/kg
and 10,8g/kg had effets on prolonging reaction time for temperature,
the difference was statistically significantly compared to the
biological control group (p < 0,05).
* The hyperalgesia to thermal stimulation in rat by the Thermal
Plantar Test Instrument: Tu dieu tan granule at the dose of 3,6g/kg
and 10,8g/kg had effets on increasing the endurance for painful force,
increasing the response time for painful stimulation, the difference
was statistically significantly compared to the biological control
group and to pre-treatment (p < 0,05).
13
3.1.4. Hypouricemic effects
* The potassium oxonat induced hyperuricemia in rat
Table 3.2. Hypouricemic effect of Tu dieu tan granule on serum uric
acid level in rats
Groups
n
Induction control group
allopurinol 20mg/kg group
10
10
Tu dieu tan granule 3,6g/kg group
10
Tu dieu tan granule 10,8g/kg
group
10
Uric acid
(mmol/L)
168,90 ± 33,59
39,40 ± 4,43∆∆∆
54,80 ±
10,04∆∆∆
50,80 ±
16,96∆∆∆
The reduction
compaired to
control group
76,67%
67,55%
69,92%
∆∆∆
Urinary u ric acid (mmo l/L)
p < 0,001 compared to the induction control group
Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had
hypouricemic effects, the difference was statistically significantly
compared to the induction control group (p < 0,001). The SUA levels
reduction at the dose 10,8g/kg higher than the dose 3,6g/kg of 2,37%.
* The xanthin oxidase inhibatory activity in vitro and in vivo
model: Tu dieu tan granule had negative result on inhibiting xanthin
oxidase reaction in vitro. In in vivo model, Tu dieu tan granule at the
dose of 2,1g/kg decreased uric acid optical absorption, the difference
was not statistically significantly compared to the control group (p >
0,05). Tu dieu tan granule had no effects on inhibiting xanthin
oxidase in vivo and in vitro.
* The diuretic and uricosuric activity in mice:
20
10
17.8
11.8
18.2
9.4
0
Group
Induction
group
Tu dieu tan
3,6g/kg
Tu dieu tan
10,8g/kg
Chart 3.1. The effects of Tu dieu tan granule on urinary serum uric
acid level in mice
Lô nghiên cứu
14
Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg increased
uric acid excretion in mice, the difference was statistically
significantly compared to the biological control group (p < 0,05).
3.2. Results of clinical studies
3.2.1. Characteristics of study subjects
A comparison between the study group and the control group
regarding age, sex, occupation, disease severity indicated (number of
swollen and painful joints, average duration of chronic gout, VAS1,
VAS2, VAS3, HAQ scores) at D0 showed no significant difference (p
> 0,05).
3.2.2. Results of supporting gout treatment in clinical and
subclinical
3.2.2.1. Supporting gout treatment in clinical and subclinical of
Tu dieu tan granule according to Western medicine
* Acute anti-inflammatory effects
Table 3.3. Improvement in the number of swollen joints
Study group
Control group
Average number of
p study-control
X ± SD (n = 60) X ± SD (n = 60)
swollen joints
D0
D30
1,05 ± 0,47
0,65 ± 0,48
1,08 ± 0,28
0,83 ± 0,37
> 0,05
< 0,05
Average
improvement D0 -
0,40 ± 0,62
0,25 ± 0,44
> 0,05
D30
p (D0 - D30)
< 0,001
< 0,001
After treatment, a statistically significant decrease in the average
number of swollen joints was found in both groups (p < 0,001). At D30,
the reductions in the study group was more pronounced than in the
control group (p < 0,05).
* Analgesic effects
Table 3.4. Improvement in the pain intensity assessed by VAS1
Control group
Study group
p study-control
VAS1 index (point)
X ± SD (n =
X ± SD (n = 60)
D0
D30
Average
improvement D0 -
6,90 ± 0,82
2,19 ± 1,10
4,71 ± 1,26
60)
6,93 ± 0,80
4,07 ± 1,03
2,87 ± 1,06
> 0,05
< 0,001
< 0,001
15
D30
p (D0-D30)
< 0,001
< 0,001
Tu dieu tan granule improved the average number of painful joints
at D30 and improved the pain intensity assessed by VAS 1 higher than
the control group and pre-treatment, the difference was statistically
significantly (p < 0,05).
* Improvement in physical activity function
Table 3.5. Improvement in physical activity function according to
VAS2, VAS3, HAQ scores
Study group
Average
Control group
p
X ± SD (n =
improvement D0 X ± SD (n = 60) study-control
D30
60)
VAS2 index (point)
4,56 ± 1,53
2,97 ± 1,12
< 0,001
VAS3 index (point)
4,58 ± 1,25
2,68 ± 1,14
< 0,001
HAQ index (point)
- 0,96 ± 0,32
- 0,42 ± 0,22
< 0,001
After treatment, both two groups had statistically significant
improvement in physical activities fucntion according to VAS2,
VAS3, HAQ scores better than pre-treatment (p < 0,001). The
improvement in the study group being more pronounced than the
control group (p < 0,001).
* Hypouricemic effects
Table 3.6. SUA levels changed in the two groups pre- and posttreatment
Study group
Control group
SUA levels
(μmol/l)
X ± SD (n = 60)
X ± SD (n = 60)
p study-control
D0
D30
521,07 ± 69,16
320,65 ± 83,88
498,18 ± 63,26
438,65 ± 95,59
> 0,05
< 0,001
Average
improvement D0
200,42 ± 100,14
57,53 ± 101,89
< 0,001
- D30
p (D0 - D30)
< 0,001
< 0,001
After treatment, both two groups had statistically significant
decreased in SUA levels more than pre-treatment (p < 0,001); the
reduction in the study group being more pronounced than the control
group (p < 0,001).
16
The percentage of patients in the study group who had the
hypouricemic treatment efficacy was 98,33%, higher than the control
group (71,67%), the effectiveness in the study group being more
pronounced than in the control group (p < 0,001).
3.2.2.2. Supporting gout treatment in clinical and subclinical of
Tu dieu tan granule according to traditional medicine
* The effect of Tu dieu tan granule on Tong Feng treatment
according to traditional medicine: Patient was assessed the Tong
Feng treatment efficacy according to Nimodiping score. After
treatment, the percentage of patients in the study group had completed
and pronounced results (98,33%) higher than the control group (15%),
the difference was statistically significantly (p < 0,001).
* The effect of Tu dieu tan granule on Tong Feng treatment in
two traditional patterns
Table 3.7. Changes in treatment evaluation indicators of two
traditional patterns in the study group
Average
improvement D0 D30
Nimodiping index
(point)
Number of swollen
joints (joint)
Number of painful
joints (joint)
VAS1 index (point)
VAS2 index (point)
VAS3 index (point)
HAQ index (point)
SUA (μmol/l)
Wind damp heat
impediment pattern
Phlegm blood
stagnation pattern
(1) X ± SD (n = 12)
(2)
X ± SD (n = 48)
P(1-2)
10,92 ± 2,31
10,62 ± 2,83
> 0,05
0,42 ± 0,51
0,39 ± 0,64
> 0,05
0,42 ± 0,52
0,44 ± 0,58
> 0,05
> 0,05
2,52 ± 0,97
2,83 ± 0,80
> 0,05
4,00 ± 1,65
4,7 ± 1,48
> 0,05
4,08 ± 1,08
4,71 ± 1,27
> 0,05
0,88 ± 0,22
1,03 ± 0,32
222,26 ± 114,84
194,97 ± 96,69 > 0,05
After treatment, the study indicators were used to evaluate the
anti-inflammatory, analgesic, hypouricemic effects, Nimodiping
score in both two traditional patterns improved better than pretreatment, the difference between two patterns was not statistically
significantly (p > 0,05).
3.3. Adverse effects
17
* In clinical: During treatment, the percentage of patients in control
group had unexpected symptoms such as digestive disorders were 5%
(3/60), itchy rash was 1,67% (1/60). Patients in the study group did
not have unexpected symptoms.
* In subclinical: There was no significant differences in the number
of red blood cells, white blood cells, platelets, hemoglobin, glucose,
triglyceride, HDL-C and laboratory tests of liver and kidney
functions, such as urea, creatinine, AST, and ALT between pre- and
post-treatment, and between the two groups. In the study group,
cholesterol and LDL-C decreased statistically significantly compared
to pre-treatment (p < 0,001).
Chapter 4
DISCUSSION
4.1. Discussion on the findings of experimental studies
4.1.1. Investigating the acute and sub-chronic toxicity effects
* Acute toxicity: The dose of 78,1g/kg was the highest dose
possible used for mices but no mices died within 72 hours, thus LD50
of Tu dieu tan granule have not been determined by oral in mices.
According to WHO guideline, Tu dieu tan granule is safety in
clinical. In the two groups which had diarrhea, mices were used Tu
dieu tan granule at the dose of 17 times clinical dose (62,5g/kg) and
21 times clinical dose (78,1g/kg). This symptom may be due to the
effect of gum in Radix Achyranthes asperae. Gum is a herbal
carbohydrate, it is not absorbed through the gastrointestinal
membranes then causes laxation, which can cause diarrhea if being
used at high dose.
* Subchronic toxicity: According to results, Tu dieu tan granule
at the dose of 1,8g/kg after 8 weeks of continuous administration and
5,4g/kg after 4 weeks of continuous administration showed no
haematopoietic toxicity and the treatment did not change the rats’
liver and kidney function indicated by biochemiscal as well as
histopathological tests. Tu dieu tan granule at the dose of 5,4g/kg
after 8 weeks of continuous administration showed the changes in
hematological indicators but still in normal standards. The bias of
reducing number of red blood cells, hemoglobin levels, and
hematocrit may be due to the action of saponin in Radix Achyranthes
18
asperae. Saponin used in high dose for long time can rupture the red
blood cells, thus affects hemoglobin level and hematocrit. This
indicator needs to be monitored in clinical studies.
4.1.2. Acute anti-inflammatory effects
* The carrageenin-induced rat paw oedema model: The results
showed that Tu dieu tan granule at the dose of 1,8g/kg and 5,4g/kg
had no anti-inflammatory effect at all times after causing
inflammation. This finding is not consistent with the previous studies
on the anti-inflammatory effects of Rhizoma Atractylodis and Radix
Achyranthes asperae on the carrageenin-induced rat paw oedema
model. Thus, we continue to conduct the rat peritonitis inflammatory
model to evaluate the mechanism of Tu dieu tan granule.
* The rat peritonitis inflammatory model: The results on table 3.1
showed that Tu dieu tan granule at both two doses reduced the volume
and protein content of rat peritonitis inflammatory statistically
significantly compared to the biological control group (p < 0,001),
reduced the number of white blood cells (p < 0,01). This result is
consistent with some studies on the anti-inflammatory effect of Radix
Achyranthes asperae, Rhizoma Atractylodis, Cortex Oroxyli, which
were found in Tu dieu tan granule component. Nguyen Huong Giang
(2014) found that the hydroxyl at position 2 of benzoxazinone in
Semen Coicis extract has anti-inflammatory effect which inhibits the
cell proliferation of nitric oxide and PGE2 by reducing the production
of nitric oxide synthetase and cyclooxygenase.
* The rat arthritis after intrasynovial injection of sodium urate:
This model was built up by Faires and McCarty which is specialize
for evaluating the anti-inflammatory effects on gouty arthritis. The
results showed that Tu dieu tan granule at the dose of 2,1g/kg had
effect on reducing knee oedema, synovial fluid, lowering the
temperature of the inflammatory sites, reducing the infiltration of
inflammatory cells after 6 hrs and 24 hours. The difference was
statistically significantly compared to the biological group (p < 0,05).
This result is consistent with the studies of Chen Guang Liang, Yang
Yan Hua which evaluated the effectiveness of Tu dieu tan formula in
the rat arthritis after intrasynovial injection of sodium urate.
Observing the images of knee pathology in rats treated Tu dieu
tan granule (image 3.1) showed that: The synovial fluid has a
19
sloughed area, however it is easy to observe. The synovial fluid had
less oedema fluid, urate crystals and infiltration of inflammed cells
than the biological control group. In particular, the urate crystals in
the synovium were rarely seen. These images showed that Tu dieu
tan granule excreted and reduced urate crystals, thereby inhibiting the
spread of the inflammatory factors. We suppose that saponin in
Radix Achyranthes asperae, polysaccharide in Rhizoma Atractylodis
and baicalein, oroxylin A in Cortex Oroxyli involved in inhibiting
gouty arthritis with different mechanisms.
4.1.3. Analgesic effects
The results in the hot plate model and the hyperalgesia to thermal
stimulation in rat by the Thermal Plantar Test Instrument showed that
Tu dieu tan granule at the dose of 3,6g/kg and 10,8g/kg had effects
on prolonging reaction time for temperature, increasing the endurance for
painful force, increasing the response time for painful stimulation, the
difference was statistically significantly compared to the biological control
group and pre-treatment (p < 0,05). The response time for painful
stimulation was prolonged shows the analgesic effects of Tu dieu tan
granule.
This effect may be due to β-eudesmol in Rhizoma Atractylodis
and other substances in Radix Achyranthes asperae. β-eudesmol has
analgesic effect in the hot plate model and the acetic acid writhing
and colorectal distention models.
4.1.4. Hypouricemic effects
* The potassium oxonat induced hyperuricemia in rat
The results on table 3.2 showed that Tu dieu tan granule at the dose of
3,6g/kg and 10,8g/kg had hypouricemic effects, the difference was
statistically significantly compared to the induction control group (p <
0,001). The SUA levels reduction at the dose 10,8g/kg higher than the
dose 3,6g/kg.
Thus, Tu dieu tan granule is effective in lowering SUA levels in
experimental. According to Liu Ku, Rhizoma Atractylodis has effective
treatment on mice with kidney failure caused by hyperuricemia.
According to Xu Jia Xin, Semen Coicis promoted the excretion of uric
acid through the urinary system, thereby reducing SUA levels.
* The xanthin oxidase inhibatory activity in vitro and in vivo
models: The results in both two models showed that Tu dieu tan
20
granule had no effects on inhibiting xanthin oxidase in vivo and in
vitro. These results suggested us to find out the other hypouricemic
mechanism of Tu dieu tan granule.
* The diuretic and uricosuric activity in mice: The results on
chart 3.1 showed that Tu dieu tan granule at the dose of 3,6g/kg and
10,8g/kg increased uric acid excretion in mice, the difference was
statistically significantly compared to the biological control group (p
< 0,05). Thus, Tu dieu tan granule had hypouricemic effects by
excreting uric acid through the urinary system. This mechanism is
related to the effects of achyranthine in Radix Achyranthes asperae
and stigmasterol, p-coumaric acid in Semen Coicis. Achyranthine
has diuretic effect in mice. This effect was demonstrated in the
Lipschitz diuretic model. Stigmasterol and p-coumaric acid
eliminate uric acid which inhibit the proliferation, aggregation of
calcium oxalate crystals, thereby inhibiting the binding of urate
crystals on epithelial cells.
According to traditional medicine, Semen Coicis has effective
on tonifying spleen qi which has been used on treating the damp
stasis pattern with numbness, difficulty in movement. Radix
Achyranthes asperae has effective on circulating blood, stimulating
meridians, diuretic, nourishing liver and kidney, dispelling wind
dampness. Radix Achyranthes asperae combined with Cortex
Oroxyli and Rhizoma Atractylodis improved the effective of Semen
Coicis on eliminating dampness, dỉuretic, analgesic. The four
herbal ingredients interacted with each other on lowering SUA
levels in experimental.
4.2. Discussion on clinical study results
4.2.1. The comparability in the characteristics of two
groups of patients
The age, gender, disease severity indicated (number of swollen
and painful joints, average duration of chronic gout, VAS1, VAS2,
VAS3, HAQ scores) were comparable between the study group and
control group. This is a major criterion in controlled experimental
studies to ensure the study objectivity.
4.2.2. Results of supporting gout treatment in clinical and
subclinical
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